Histone H1 Kinase Activity Assay

Protein lysates were prepared as described by Hlavová et al. [32 (link)]. They were directly assayed or affinity-purified by CrCKS1 beads as described by Bisova et al. [19 (link)] with modifications as described by Hlavová et al. [32 (link)]. Histone H1 kinase activity was assayed as previously described [33 (link)] in a final volume of 10 µL with either 7 µL of clear whole cell lysate or the CrCKS1 beads fraction corresponding to 20 µL of whole cell lysate. The reactions were initiated by adding the master mix to a final composition of 20 mM HEPES, pH 7.5, 15 mM MgCl₂, 5 mM EGTA, 1 mM DTT, 0.1 mM ATP, 0.2% (w/v) histone (Sigma H5505) and 0.370 MBq [γ ³²P] ATP. All the chemicals were purchased from Sigma-Aldrich (Prague, Czech Republic).
Proteins were separated on 15% SDS-PAGE gels [34 (link)]. Phosphorylated histone bands were visualized by autoradiography, analyzed using a Phosphoimager (Storm 860, Molecular Dynamics, GE Healthcare, Prague, Czech Republic), and quantified using Image Studio Lite software (LI-COR Biosciences, v. 5.2, Lincoln, NE, USA) as described by Zachleder et al. [4 (link)].